Transplantation of Neural Stem Cells
The CNS has a very limited regenerative capacity. Thus it is of major interest to investigate the ability of human neural stem cells (NSCs) engrafted into the brain to survive, migrate and integrate in a functional and meaningful manner.
Studies have shown that stem cells derived from the embryonic or fetal human brain can be successfully grafted into the developing rodent CNS. Once transplanted, these cells survive, migrate and integrate into the host tissue, giving rise to cells from the three fundamental neuronal lineages i.e. neurons, astrocytes and oligodendrocytes (Brustle et al., 1998; Flax et al., 1998; Uchida et al., 2000; Englund et al., 2002b; Peng et al., 2002; Honda et al., 2007).
However, transplantation studies in the adult CNS are more challenging. As the tissue is fully established, developmental cues are limited and space is more constricted (Svendsen & Caldwell, 2000) leading to restricted migration and integration of the transplanted cells. Engraftment of fetal or ES cell derived neural progenitors in the adult CNS could show that transplanted cells survive but form a graft core meaning that the majority of the transplanted cells remain mainly situated at the grafted site (Guzman et al., 2008). Restricted migration of the transplanted cells could be observed 10 to 15 weeks following engraftment (Fricker et al., 1999; Aleksandrova et al., 2002; Englund et al., 2002a; Tabar et al., 2005; Roy et al., 2006; Guzman et al., 2008). It was suggested that physical or molecular barriers caused by glial scarring at the transplantation site are the reason for the restricted outgrowth of transplanted cells (Reier et al., 1983; Rudge & Silver, 1990). Such effect might be solved by microtransplants, which minimize scarring at the grafted site (Nikkhah et al., 1995; Davies et al., 1997).
Nevertheless, cell replacement therapies for diseases of the adult brain have attracted attention since the first reports of successful transplantation of embryonic dopaminergic cells to patients with Parkinson's disease (Lindvall & Hagell, 2001). Parkinson's disease is characterized by a loss of dopamine-producing midbrain neurons with cell bodies in the substantia nigra. These neurons project to the striatum and are essential for motor function. Parkinson's patients suffer from various symptoms including resting tremor, difficulty in walking, and loss of facial expression. The disease is typically progressive due to ongoing loss of neurons. The first transplantation studies with fetal tissue in animal models of Parkinson's disease have shown that grafted dopaminergic cells are able to release dopamine at near normal levels and that the animals show significant behavioural recovery (Annett et al., 1994; Herman & Abrous, 1994; Lindvall et al., 1994). Positive effects have also been observed in clinical trails with human patients (Olanow et al.; Lindvall, 1999). Major improvements, however, were only seen in patients aged 60 years or younger (Freed et al.). Moreover, some patients receiving transplants developed dyskinesias, movement disorders associated with excessive dopamine levels in the brain. Further success of these transplantation approaches has been constrained by limited availability of fetal tissue, limited migration of grafted cells, and poor differentiation and survival of the grafted neurons (Richardson et al., 2004). In addition to these problems, fresh fetal tissue cannot be standardized and raises ethical questions that have been debated intensely (Bjorklund & Lindvall, 2000).
Many of these issues can be better addressed by working towards an in vitro culture system. The knowledge about hES cells, including techniques of producing stable well-characterised NSCs from hES cells has provided prospects to generate large numbers of donor cells for neural repair (Koch et al., 2009). Many studies already show that neural progenitors derived from ES cells can give rise to dopaminergic neurons. This is mainly achieved by the combined use of FGF8 and Shh, which effectively induce dopaminergic neurons from ES cell-derived neural progenitors (Lee et al., 2000; Yan et al., 2005). Addition of ascorbic acid, brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), dibutyryl cyclic-AMP, and transforming growth factor-beta 3 (TGF-β3) yields cultures containing 30% to 50% neurons expressing beta-III tubulin, of which 65% to 80% express tyrosine hydroxylase required for dopamine synthesis. These neurons release dopamine upon depolarization, and form at least rudimentary synapses in vitro with transmitter re-uptake abilities (Kim et al., 2007; Joannides et al., 2007). Following transplantation these cells survive, maintain their dopaminergic phenotype and functionally engraft in the brain (Sanchez-Pernaute et al., 2005; Yang et al., 2008). Using cultured ES cell-derived neural precursors as a source for transplantation therapies may, on the one hand, obviate some of the technical limitations associated with the use of fresh fetal tissue (Ostenfeld & Svendsen, 2003), but may also on the other hand, bear the risk of teratoma formation. Currently, the only way to ensure that teratomas do not form is to differentiate the ES cells in advance, enrich for the desired cell type and screen for the presence of undifferentiated cells. In addition, hES cell-derived neural precursor transplants have been found to give rise to proliferating neural clusters rather than individually incorporating neurons (Roy et al., 2006) indicating that even committed progenitors can proliferate excessively after transplantation. This problem might be solved by using more restricted precursor cells or by the purification of desired postmitotic subtypes of neurons or glia.
Compared to cell replacement therapy for Parkinson's disease, in which one specific type of neurons has to be replaced by a direct local cell transplantation, cell therapy for stroke or spinal cord injury is a major challenge as transplanted NSCs need to replace a range of neuronal types, remyelinate axons and repair complex neural circuitries. In addition, it is required that transplanted cells reach the lesion site by following a gradient of inflammatory cues such as cytokines and chemokines (Ransohoff, 2002). As a preliminary step towards this goal, it was shown that human NSCs transplanted into the brains of rodents after stroke survived, migrated, and differentiated into various types of neurons (Aoki et al., 1993; Ben-Hur et al., 2003; Imitola et al., 2004; Kelly et al., 2004). Other degenerative diseases of the adult CNS such as Alzheimer's disease and amyotrophic lateral sclerosis would also require the migration of transplanted cells towards specific sites within the CNS. Many neurodegenerative diseases are associated with a non-permissive environment, which can inhibit regenerative processes. These circumstances create an even bigger challenge for cell replacement therapy.
Thus, the major difficulties yet to be solved are how to direct and control the differentiation of specific phenotypes required for replacement and repair in each disease, how to purify lineage specific subtypes and how to improve cell migration and integration into the affected site of the CNS.
Several previous studies analysed the migration and integration potential of hES cell-derived neurons in vivo. Former studies, in which hES cell-derived neural cells were transplanted into rat brains, described clusters of donor cells at the site of engraftment one week after transplantation, the so called transplantation cores (Reubinoff et al., 2001). Tabar (Tabar et al., 2005) and co-workers investigated in vivo migration of hES cell-derived neural precursors transplanted into the rostral migratory stream of adult rats and found that about one fourth of the transplanted cells migrated out of the transplantation core within 11 weeks.
In comparison, the inventors observed when transplanting pure populations of immature human neurons into the striatum of adult rats, a large amount of the cells migrated out of the transplantation core within 8 days. Similar results have been achieved following transplantation into the rostral migratory stream, where transplanted neurons morphologically orientated to and migrated towards the olfactory bulb within 8 days. In contrast, the corresponding immature neurons within a cell mixture with neural stem/progenitor cells did not show such a strong migratory behaviour although they should in principle have the same migration potential as the pure neurons. It was observed that the cells of mixed neural/neuronal transplants formed densely packed clusters at the transplantation site with only restricted migration of neurons out of the transplantation core.
Cluster formation and limited migration and integration have been topics in neurobiological research for many years. A major challenge in therapeutic transplantation of donor cells for neural damage repair is to achieve functional integration of the donor cells into the host tissue. Limited integration due to restricted emigration of the transplanted cells, which mainly remain located at the grafted site (Guzman et al., 2008) is a widely discussed issue and described in many different studies using primary cells or ES cell derived neural progeny (Fricker et al., 1999; Tabar et al., 2005; Roy et al., 2006). It is argued that this core formation of neural transplants is due to physical or molecular barriers caused by glial scarring at the lesion site following transplantation (Reier et al., 1983; Rudge & Silver, 1990). Successful axonal outgrowth is known to be associated with minimal up-regulation of proteoglycans within the extracellular matrix of reactive glial cells at the transplantation site (Davies et al., 1997). This might also restrict migration of transplanted neuronal progenitors. It was suggested that the up-regulation of proteoglycans might be avoided by using microtransplants that minimize scarring by injecting minimal volumes of dissociated cells (Nikkhah et al., 1995; Davies et al., 1997).
Erythropoietin
Erythropoietin (EPO) is a member of the hematopoietic growth factor family and behaves as a hormone. It is responsible for the regulation of red blood cell (erythrocyte) production (erythropoiesis), maintaining the body's red blood cell mass at an optimum level. EPO production is stimulated by reduced oxygen content in the renal arterial circulation, mediated by a transcription factor that is oxygen-sensitive. EPO is a produced primarily by cells of the peritubular capillary endothelium of the kidney. Secreted EPO binds to EPO receptors on the surface of bone marrow erythroid precursors, resulting in their rapid replication and maturation to functional red blood cells. This stimulation results in a rapid rise in erythrocyte counts and a consequent rise in hematocrit (% of red blood cells in blood) (D'Andrea et al Cell 1989 57: 277-285. Lodish et al Cold Spring Harb Symp Quant Biol 1995 60: 93-104).
Human EPO was first cloned and amino acid sequence reported by Lin et al. (Proc. Natl. Acad. Sci. USA 1985 82: 7582-4) and Jacobs K. et al. (Nature 313: 806-810 1985).
Human EPO is an acidic glycoprotein with a molecular weight of approximately 30400 daltons. It is composed of an invariant 165 amino acid single polypeptide chain containing four cysteine residues (at positions 7, 29, 33 and 161), which form the internal disulphide bonds (Lai et al., J. Biol. Chem. 1986, 261: 3116-3121; Recny et al. J. Biol. Chem. 1987 262: 17156-17163). The disulphide bridge between cysteine 7 and 161 is known to be essential for biological activity. The carbohydrate portion of EPO consists of three N-linked sugars chains at Asn 24, 38 and 83, and one O-linked sugar at Ser 126 (Browne J. K. et al. Cold spring Harb. symp. Quant. Biol. 1986, 51: 693-702 Egrie J. C. et al. Immunbiology 1986 172: 213-224.)
The structure of human EPO has been reported (Cheetham et al 1988 Nat. Struct. Biol. 5:861-866; Syed et al. 1998 Nature 395:511-516). Human EPO is a four helix bundle, typical of members of the hematopoietic growth factor family. In contrast to the invariant amino acid sequence, the carbohydrate structures are variable, a feature referred to as micro-heterogeneity. The differences in carbohydrate moieties, in terms of the branching pattern, complexity size and charge have profound effects on the pharmacokinetics and pharmacodynamics of EPO. The effects of different glycosylation patterns have been well studied (Darling et al. 2002 Biochemistry 41: 14524-14531; Storring et al. 1998 Br. J. Haematol. 100: 79-89; Halstenson et al 1991 Clin. Pharmacol. Ther. 50: 702-712; Takeuchi et al. 1990 J. Biol. Chem. 265: 12127-12130).
The following EPOs have the same amino acid sequence as recombinant human EPO (rhEPO) and variations in the methods of production and glycosylation distinguish these products. Epoetin alfa (genomic DNA) and epoetin beta (cDNA) are described in U.S. Pat. Nos. 4,703,008 and 5,955,422. These have the same amino acid sequence as human EPO and are produced in chinese hamster ovary (CHO) cells. Epoetin alfa is available under the trade names procrit (Ortho Biotech), eprex (Johnson & Johnson), epogin (Chugai) or epogen (Amgen). Epoetin beta is available under the trade name neorecormon or recormon (Hoffmann-La Roche). It was developed by the Genetics Institute for the treatment of anaemia associated with renal disease. Epoetin omega described in U.S. Pat. No. 5,688,679 has the same amino acid sequence as human EPO and is produced in baby hamster kidney cells (BHK-21). Epoetin omega is available under the trade names EPOMAX (Elanex).
Darbepoetin alfa (novel erythropoiesis stimulating protein, NESP) was developed by Amgen and is available under the trade name ARANESP (Macdougall I. C., Kidney Int. Suppl. 2002 May; (80):55-61). It was designed to contain five N-linked carbohydrate chains (two more than rhEPO). The amino acid sequence of Aranesp differs from that of rhEPO at five substitutions (Ala30Asn, His32Thr, Pro87Val, Trp88Asn, Pro90Thr), thus allowing for additional oligosaccharide attachment at asparagine residues at position 30 and 88. Due to its increased carbohydrate content, Aranesp differs from rhEPO as a result of a higher molecular weight (37,100 compared to 30,400 Daltons), sialic acid content (22 compared to 14 sialic acid residues) and increased negative charge. The increased carbohydrate content of Aranesp accounts for its distinct biochemical and biological properties, in particular a 3-fold longer circulating half-life than other existing erythropoietins when administered via the intravenous (IV) or subcutaneous (SC) route. However, the relative EPO receptor binding affinity was inversely correlated with the carbohydrate content, with Aranesp displaying a 4.3-fold lower relative affinity for the EPO receptor than that of rhEPO. Following SC administration, the absorption of Aranesp is slow and rate-limiting, serum levels reaching a maximum at a mean of 54 h. The time to maximum concentration is longer than that reported for rhEPO, probably because of the increased molecular size of Aranesp. However currently, the extended circulating half-life gives Aranesp a significant clinical advantage over Procrit due to its less frequent dosing. Opportunities may exist however, to explore possible improvements to the affinity of Aranesp for its receptor or to address the rate of absorption following SC administration.
Transkaryotic Therapies (in conjunction with Aventis Pharma) are developing erythropoietin stimulant Dynepo (epoetin delta). Dynepo is a gene-activated human erythropoietin produced in human cell culture, for the treatment of anemia in patients with renal failure.
Roche is developing R-744, continuous erythropoietin receptor activator (CERA), a second-generation erythropoietin, for the potential treatment of anemia associated with chemotherapy. CERA contains a single methoxypolyethylene glycol polymer of approximately 30 kDa that extends the half life of this agent.
Many EPO individual point mutants have been made to study the EPO structure activity relationship (Elliot et al. 1997 Blood 89: 493-502; Elliot et al. 1996 Blood 87: 2702-2713; Syed et al. 1998 Nature 395: 511-516) or effects of glycosylation (O'Narhi et al. 2001 Protein Engineering 14: 135-140; Bill et al. 1995 Biochimica et Biophysica Acta 1261: 35-43; Yamaguchi et al. 1991 J Biol Chem 266: 20434-20439).
EPO is a major biopharmaceutical product with world-wide sales topping US$ 3 billion. It is used primarily to boost erythrocyte and red blood cell formation in patients to treat anaemia associated with chronic renal failure, cancer chemotherapy, HIV infection, pediatric use, premature infants and to reduce the need for blood transfusions in anaemic patients undergoing elective non-cardiac and non-vascular surgery.
Endostatin
Endostatin is a 20 kDa C-terminal fragment of collagen XVIII, a member of a family of collagen-like proteins called multiplexins (O'Reilly, M. S. et al. Endostatin: An endogenous inhibitor of angiogenesis and tumor growth. Cell 1997, 88: 277-285). Collagen XVIII is a component of the basement membrane zones that surround blood vessels (Muragaki, Y. et al. Mouse col18a1 is expressed in a tissue-specific manner as three alternative variants and is localized in basement membrane zones. Proc. Natl. Acad. Sci. USA 1995, 92, 8763-8767). Endostatin is an inhibitor of angiogenesis. It specifically inhibits endothelial cell proliferation, that is, it has no effect on the growth of other cell types. It is produced naturally by a murine hemangioendothelioma, from which it was first purified (O'Reilly, M. S. et al., supra). Recombinant E. coli-derived endostatin, when added at a site remote from the primary tumor, has a systemic effect causing even very large tumors (1% of body weight) to regress to dormant microscopic nodules (O'Reilly, M. S. et al., supra). Hence tumors can be forced to regress over 150-fold in size to less than 1 mm3. As long as treatment is continued there is no tumor regrowth, and no toxicity. When treatment is initially stopped tumors regrow, however treatment can be continued and drug-resistance does not develop over multiple treatment cycles (Boehm, T. et al., Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 1997, 390: 404-407). Remarkably, repeated cycles of antiangiogenic therapy were followed by self-sustained dormancy that remained for the lifetime of most animals (Boehm et al., supra). The mechanism for the persistence of tumor dormancy after therapy is suspended is unknown, but it is not due to an antitumor immune response, as tumors injected at sites remote from the treated tumor grew unchecked. The dormant tumors which are of a size that can survive without blood vessels display no net gain in size due to a balance between high proliferation of tumor cells, and high apoptosis.
The mechanism of action of endostatin remains unknown. The anti-angiogenic effects of endostatin may be due in part to its ability to block the attachment of endothelial cells to fibronectin via α5β1, and αVβ3 integrins (Rehn, M. et al., Interaction of endostatin with integrins implicated in angiogenesis. Proc. Natl. Acad. Sci. USA 2001, 98: 1024-1029) and/or α2β1 (Furumatsu, T. et al., Endostatin inhibits adhesion of endothelial cells to collagen I via alpha(2)beta(1) integrin, a possible cause of prevention of chondrosarcoma growth. J. Biochem. 2002, 131: 619-626.).
Angiostatin
Angiostatin is a 38,000-Mr protein comprising the first four of five highly homologous 80-amino acid residue long triple-loop structures termed kringles (O'Reilly M. S. et al., Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell. 1994; 79:315-328). It can inhibit the growth of a broad array of murine and human tumors established in mice (O'Reilly M. S. et al., Angiostatin induces and sustains dormancy of human primary tumours in mice. Nat. Med. 1996; 2:689-692), and is non-toxic such that tumors can be subjected to repeated treatment cycles, without exhibiting acquired resistance to therapy (Boehm T et al., supra). Its tumor-suppressor activity may arise from its ability to inhibit the proliferation of endothelial cells by binding to the α/β-subunits of ATP synthase (Moser T. L. et al., Angiostatin binds ATP synthase on the surface of human endothelial cells. Proc Natl Acad Sci USA 1999; 96:2811-2816), by inducing apoptotic cell death (Holmgren L. et al., Dormancy of micrometastases-balanced proliferation and apoptosis in the presence of angiogenesis suppression. Nat. Med. 1995; 1: 149-153), by subverting adhesion plaque formation and thereby inhibiting the migration and tube formation of endothelial cells (Claesson-Welsh L. et al. Angiostatin induces endothelial cell apoptosis and activation of focal adhesion kinase independently of the integrin-binding motif RGD. Proc Natl Acad Sci USA. 1998; 95:5579-5583), and/or by down-regulating vascular endothelial growth factor (VEGF) expression (Kirsch M. et al. Angiostatin suppresses malignant glioma growth in vivo. Cancer Res. 1998; 58:4654-4659; Joe Y. A. et al. Inhibition of human malignant glioma growth in vivo by human recombinant plasminogen kringles 1-3. Int. J. Cancer 1999; 82:694-699). Angiostatin reduces the phosphorylation of the mitogen-activated protein kinases ERK-1 and ERK-2 in human dermal microvascular cells in response to VEGF (Redlitz A. et al. Angiostatin diminishes activation of the mitogen-activated protein kinase ERK-1 and ERK-2 in human dermal microvascular endothelial cells. J. Vasc. Res. 1999; 36:28-34). Endothelial progenitor cells are exquisitively sensitive to the effects of angiostatin, and may be the most important target of angiostatin (Ito H. et al. Endothelial progenitor cells as putative targets for angiostatin. Cancer Res. 1999; 59:5875-5877). Gene transfer of angiostatin into small solid EL-4 lymphomas established in mice led to reduced tumor angiogenesis, and weak inhibition tumor growth (Sun, X. et al. Angiostatin enhances B7.1-mediated cancer immunotherapy independently of effects on vascular endothelial growth factor expression. Cancer Gene Ther. 8: 719-727, 2001). In contrast, when angiostatin gene therapy was preceded by in situ gene transfer of the T cell costimulator B7-1, large tumors were rapidly and completely eradicated; whereas B7-1 and angiostatin monotherapies were ineffective. Gene transfer of AAV-angiostatin via the portal vein led to significant suppression of the growth of both nodular and, metastatic EL-4 lymphoma tumours established in the liver, and prolonged the survival time of the mice (Xu, R. et al. Long-term expression of angiostatin suppresses metastatic liver cancer in mice. Hepatol. 37:1451-60, 2003). Survivin is a recently identified member of the inhibitor of apoptosis (IAP) proteins (Ambrosini, G. et al. 1997. A novel anti-apoptosis gene. Survivin expression in cancer and lymphoma. Nat. Med. 3: 917-921) which are now regarded as important targets in cancer therapy. Antisense complementary DNA (cDNA) and oligonucleotides that reduce the expression of the IAP protein Bcl-2 inhibit the growth of certain tumor cell lines in vitro (Ambrosini et al. 1997, supra; Webb, A. et al. 1997. BCL-2 antisense therapy in patients with non-Hodgkin lymphoma. Lancet 349:1137-1141; Miayake, H. et al. 2000. Chemosensitization and delayed androgen-independent recurrence of prostate cancer with the use of antisense Bcl-2 oligodeoxynucleotides. J. Natl. Cancer Inst. 92: 34-41). Similarly, antisense oligonucleotides that reduce survivin expression in tumors cells induce apoptosis and polyploidy, decrease colony formation in soft agar, and sensitize tumor cells to chemotherapy in vitro (Baba, M. et al., 2000. In vivo electroporetic transfer of Bcl-2 antisense oligonucleotide inhibits the development of hepatocellular carcinoma in rats. Int. J. Cancer 85: 260-266; Li, F. Z. et al. 1999. Pleiotropic cell-division defects and apoptosis induced by interference with survivin function. Nat. Cell Biol. 1: 461-466; Chen, J. et al. 2000. Down-regulation of survivin by antisense oligonucleotides increases apoptosis, inhibits cytokinesis and anchorage-independent growth. Neoplasia 2:235-241; Grossman, D. et al. 1999. Expression and targeting of the apoptosis inhibitor, survivin, in human melanoma. J. Invest. Dermatol. 113:1076-1081). Intratumoral injection of plasmids that block survivin expression were found to inhibit tumor growth, particularly the growth of large tumors (Kanwar, J. R. et al. 2001. Effect of survivin antagonists on the growth of established tumors and B7.1 immunogene therapy. J. Natl. Cancer Inst. 93:1541-1552.).
Vascular Endothelial Growth Factor (VEGF) and their Receptors
VEGF was identified as a protein that induces proliferation and migration of endothelial cells in vitro, and blood vessel permeabilization and angiogenesis in vivo. It regulates both vascular proliferation and permeability. Also known as vascular permeability factor (VPF), it is unique among pro-angiogenic factors because of its specificity for vascular endothelium and potency. It also functions as an anti-apoptotic factor for endothelial cells in newly formed vessels. VEGF is expressed in tumor cells, macrophages, T cells, smooth muscle cells, kidney cells, mesangial cells, keratinocytes, astrocytes, and osteoblasts.
The VEGF family comprises seven members, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placental growth factor (PlGF). All of them have a common structure of eight cysteine residues in a VEGF homology domain. In addition, in relation to VEGF-A, there are six different isoforms, and VEGF-A165 is the main isoform. All these isoforms have distinct and overlapping functions in angiogenesis. The VEGF gene is located on chromosome 6p. 21. The different members of VEGF family have different physical and biological properties and they act through specific tyrosine kinase receptors (VEGFR-1 (also termed Flt-1), VEGFR-2 (also termed Flk-1/KDR), and VEGFR-3 (also termed (Flt-4)). The VEGFR-3 receptor and its ligands, VEGF-C and VEGF-D, are associated with lymphangiogenesis, while PlGF is linked to arteriogenesis.
A synthetic peptide, ATWLPPR has been shown to abolish VEGF binding to cell-displayed KDR, and abolished VEGF-induced angiogenesis in a rabbit corneal model (Binetruy-Tornaire, R. et al., Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis. EMBO J. 2000, 19:1525-1533).
Platelet-Derived Growth Factors (PDGF) and their Receptors
The PDGF family comprises PDGF-A, -B, -C and -D, which form either homo- or heterodimers (PDGF-AA, -AB, -BB, -CC, -DD. The four PDGFs are inactive in their monomeric forms. The PDGFs bind to the protein tyrosine kinase receptors PDGF receptor-α and -β. These two receptor isoforms dimerize upon binding the PDGF dimer, leading to three possible receptor combinations, namely -αα, -ββ and -αβ. The extracellular region of the receptor consists of five immunoglobulin-like domains while the intracellular part is a tyrosine kinase domain. The ligand-binding sites of the receptors are located to the three first immunoglobulin-like domains. PDGF-CC specifically interacts with PDGFR-αα and -αβ, but not with -ββ, and thereby resembles PDGF-AB. PDGF-DD binds to PDGFR-ββ with high affinity, and to PDGFR-αβ to a markedly lower extent and is therefore regarded as PDGFR-ββ specific. PDGF-AA binds only to PDGFR-αα, while PDGF-BB is the only PDGF that can bind all three receptor combinations with high affinity.
Dimerization is a prerequisite for the activation of the kinase. Kinase activation is visualized as tyrosine phosphorylation of the receptor molecules, which occurs between the dimerized receptor molecules (transphosphorylation). In conjunction with dimerization and kinase activation, the receptor molecules undergo conformational changes, which allow a basal kinase activity to phosphorylate a critical tyrosine residue, thereby “unlocking” the kinase, leading to full enzymatic activity directed toward other tyrosine residues in the receptor molecules as well as other substrates for the kinase. Expression of both receptors and each of the four PDGFs is under independent control, giving the PDGF/PDGFR system a high flexibility. Different cell types vary greatly in the ratio of PDGF isoforms and PDGFRs expressed. Different external stimuli such as inflammation, embryonic development or differentiation modulate cellular receptor expression allowing binding of some PDGFs but not others. Additionally, some cells display only one of the PDGFR isoforms while other cells express both isoforms, simultaneously or separately.
FGF2
FGFs are a family of polypeptides synthesized by a large number of cells during embryonic development and by cells of adult tissues under various pathological conditions.
FGF2 (or b-FGF) is the first and the most well-characterized of these growth factors. FGF2 is an 18 kDa protein which induces proliferation, migration and protease production by endothelial cells in culture and neovascularization in vivo. FGF2 interacts with endothelial cells by means of two classes of receptors, high-affinity receptor tyrosine kinases (FGFRs) and low-affinity heparan sulphate proteoglycan (HSPG) type receptors located at the cell surface and in extracellular matrices. Thus, FGF2 and its receptors represent very relevant targets for therapies aimed at activating or inhibiting angiogenic processes.
BIBF1120
Small-molecule tyrosine kinase inhibitors (RTKIs) represent a new class of targeted drugs in oncology. The RTKI BIBF1120 is a novel indolinone derivative that simultaneously and potently inhibits VEGF receptors 1 to 3 (VEGFR), PDGFR α and β as well as FGFR 1 to 3 tyrosine kinases with low cross-reactivity against a panel of other kinases (Kulimova et al.; Hilberg et al., 2008; Roth et al., 2009). BIBF1120 is thought to bind to the ATP binding pocket of the kinase domain, thereby interfering with the cross-autophosphorylation of the receptor homodimers. Its function was studied in three cell types contributing to angiogenesis: endothelial cells, pericytes and smooth muscle cells. In these cells BIBF1120 was shown to inhibit the mitogen-activated protein kinase (MAPK) and Akt signalling pathways, resulting in the inhibition of cell proliferation and apoptosis (Kulimova et al., 2006). A distinct pharmacodynamic feature of BIBF1120 in cell culture is the sustained pathway inhibition (up to 32 hours after 1-hour treatment), suggesting slow receptor off-kinetics. In all tumor models tested so far, BIBF1120 is highly active at well-tolerated doses. Although BIBF1120 is rapidly metabolized in vivo by methylester cleavage, resulting in a short mean residence time, once daily oral dosing is fully efficacious in xenograft models (Chaudhary et al., 2007; Hilberg et al., 2008). After passing phase I and II clinical studies in patients with advanced solid tumors (du Bois et al.; Mross K B, 2005; Von Pawel & Gatzemeier, 2007; Roth et al., 2009), BIBF1120 is now in phase III clinical trials.